Novel cell epitopes and combination of cell epitopes for use in the immunotherapy of myeloma  and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer, in particular myeloma. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 15/191,895, filed Jun. 24, 2016, which claimspriority from GB 1511191.7 filed Jun. 25, 2015 and U.S. ProvisionalApplication No. 62/184,500 filed Jun. 25, 2015. Each of theseapplications is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-049002_Sequence_Listing_ST25.txt” createdon 16 Nov. 2018, and 38,243 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I and II molecules of human tumorcells that can be used in vaccine compositions for eliciting anti-tumorimmune responses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Multiple myeloma (MM), a low-grade B cell lymphoma, is characterized bythe proliferation of malignant plasma cells in the bone marrow [14].Despite recent advances in treatment, including high-dose chemotherapyfollowed by autologous stem cell transplantation, novel immunomodulatorydrugs and proteasome inhibitors, MM remains largely incurable [15, 16].This is mostly due to the persistence of minimal residual disease (MRD),which leads to high relapse rates [17, 18].

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and myeloma in particular. There is alsoa need to identify factors representing biomarkers for cancer in generaland myeloma in particular, leading to better diagnosis of cancer,assessment of prognosis, and prediction of treatment success.

Antigen-specific immunotherapy holds the potential to induce clinicallyeffective anti-cancer T-cell responses and might be harnessed to guideand increase the specificity of cancer immunotherapy in futurecombination trials [3]. To this end, the exact knowledge oftumor-associated/specific T-cell epitopes is crucial. After decades ofresearch into overexpressed tumor antigens, more recently the focus hasshifted to the patient-individualized identification of mutation-derivedneoantigens [4, 5]. The encouraging findings of these new studies [6-8]have led to neoepitopes being viewed as the dominant targets ofanti-cancer immune responses [9-11].

However, analyzing the antigenome of hematological malignancies, theinventors have recently demonstrated that non-mutated antigens arerelevant targets of spontaneous anti-leukemia T-cell responses [12, 13].The strategy implemented in these studies differentially maps thenaturally presented HLA ligandomes of hematological cells in health anddisease by mass spectrometry and was found to efficiently identifyrelevant tumor-associated antigens.

So far, the only established immunotherapeutic approach for MM isallogenic stem cell transplantation, which is associated with a highmorbidity and mortality and remains an option for only a fraction ofpatients [19-21]. Antigen-specific T-cell based immunotherapy [22,23]—especially in the constellation of MRD characterized by favorableeffector to target ratios—might present an effective, low side effectoption [24].

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anti-cancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed. Complexesof peptide and MHC class I are recognized by CD8-positive T cellsbearing the appropriate T-cell receptor (TCR), whereas complexes ofpeptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell-(CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated peptides of the invention can act as MHC class II activeepitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

An array of myeloma-associated T-cell antigens has been described inprevious studies [25-35]. Most of these antigens were identified basedon gene expression analysis and reverse immunology. Some of theseantigens (WT1 [36, 37], RHAMM [38, 39], hTERT [40] and Survivin [40,41]) have already found their way into clinical trials, showingpromising results in terms of induction of specific T-cell responses aswell as clinical responses in single patients. However, broad clinicaleffectiveness has not yet been achieved. These previous studies wererestricted to very few HLA-allotypes and single antigens/epitopes [42],limiting both, the population of patients eligible for this therapeuticapproach and the spectrum of inducible tumor-specific T-cell responses.Of note, recent studies demonstrated lacking degrees oftumor-association for several of these tumor antigens, both on thetranscriptome level [43] and importantly also on the level of HLArestricted presentation [12, 13].

Kowalewski et al. (in: Kowalewski et al. Carfilzomib alters theHLA-presented peptidome of myeloma cells and impairs presentation ofpeptides with aromatic C-termini. Blood Cancer J. 2016 Apr. 8) disclosethat multiple myeloma is an immunogenic disease, which might beeffectively targeted by antigen-specific T-cell immunotherapy. Therelative presentation levels of 4780 different HLA ligands werequantified in an in vitro model employing carfilzomib treatment of MM.1Sand U266 myeloma cells, which revealed significant modulation of asubstantial fraction of the HLA-presented peptidome. These findingsimplicate that carfilzomib mediates a direct, peptide motif-specificinhibitory effect on HLA ligand processing and presentation. As asubstantial, and this may have broad implications for the implementationof antigen-specific treatment approaches in patients undergoingcarfilzomib treatment.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens. It is therefore anobject of the present invention, to provide novel epitopes to be used inthe immunotherapy of cancer, in particular of myeloma.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 228 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 228, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-6B depict embodiments as described herein.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

By analyzing the antigenic landscape of MM directly on the HLA ligandlevel the inventors here provide a panel of novel myeloma-associatedepitopes suited for antigen-specific immunotherapy.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 228 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 228,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, the HLA binding, and theprospective source (underlying) genes for these peptides.

TABLE 1 Peptides according to the present invention SEQ ID No. sequenceHLA Gene name   1 AASPVVAEY A*24 LIME1   2 AENAPSKEVL B*40 SLC1A5   3AEQEIARLVL B*40: 01 CREB3   4 AFIQAGIFQEF A*23: 01 RAD1   5 AHSEQLQALB*39 TXNDC11   6 AIILEAVNLPVDH class II SLC1A5   7 AKRFDVSGY B*15 PDIA4  8 ALDPLADKILI A*02: 01 CRLS1   9 ALKKPIKGK A*03 SETD8  10 ALWGRTTLKA*03 DAP3  11 APFQGDQRSL B*07 IRF9  12 APKYGSYNVF B*42: 01 MOGS  13APRHPSTNSL B*07 NDUFAF4  14 APRHPSTNSLL B*07 NDUFAF4  15 APVGlmFLVAGKIVclass II SLC1A5  16 APVGlmFLVAGKIVE class II SLC1A5  17 ASNPSNPRPSKA*30: 01 WHSC1 1  18 AVFIAQLSQQSLDF class II SLC1A5  19 DALGAGILHHL A*02SLC1A4  20 DEVLLQKL B*18 PPP2R3C  21 DGDDVIIIGVFKGESDPAY class II PDIA4 22 DIKDTDVlmKR A*33 MB21D1  23 DIQDPGVPR A*33 SEMA4A  24 DLFRYNPYLKRA*03 NBN  25 DLLDGFIAR A*03 CRLS1  26 DLNFPEIKR A*03 NOC2L  27DLRPATDYHVR A*33 FNDC3B  28 DRYLLGTSL B*27 ASS1  29 DSFERSNSL A*68: 02TBC1D4  30 DTQSGSLLFIGR A*03 SERPINH1  31 DVAEPYKVY A*25 IRF9  32DVNNIGKYR A*03 LAP3  33 DVPDHIIAY A*03 KIAA1217  34 EGNPLLKHYRGPAGDAclass II SCL1A5  35 EGNPLLKHYRGPAGDAT class II SLC1A5  36 EIIEKNFDY A*26TMEM126B  37 EIIEKNFDYLR A*03 TMEM126B  38 EITEVALEY A*26 TXNDC11  39ENGVLVLNDANFDNFV class II PDIA4  40 EQLYDLTLEY B*39 NOC2L  41 ERFEKTFQLB*39 MOGS  42 EYGHIPSF A*24: 02 ARHGAP11A  43 FAQIISVALI A*02 DOLK  44FAYPAIRYL A*02 DAP3  45 FFKPHWDEKF A*24 SERPINH1  46 FISGHTSEL A*02 MOGS 47 FKSPAASSF B*15 NUPL2  48 FLFQLLQLL A*02 SEMA4A  49 FLWDEGFHQLA*02: 01 MOGS  50 FNFLRNVSL B*08: 01 ARHGAP11A  51 FVFPGELLL A*02: 01SLC1A5  52 GAKASTTSL C*03: 03 CMTR1  53 GELIEVVHL B*40 NUDT14  54GETAFAFHL B*40: 01 SLX1A  55 GEVAPSMFL B*40: 01 NPC1  56 GEVQDLLVRL B*40BAZ2B  57 GKVQENSAY B*15 NOC2L  58 GKYIFASIL B*15 SLC1A4  59GNPLLKHYRGPAGDA class II SLC1A5  60 GPFSQFIKA B*55 FNDC3B  61 GPRPITQSELB*07 UBL7  62 GRYPGVSNY B*27 NAE1  63 GYPTIKILKKGQAVDYE class II PDIA4 64 GYPTIKILKKGQAVDYEG class II PDIA4  65 HPKQPEPSA B*42: 01 TXNDC11  66HPKQPEPSAT B*42: 01 TXNDC11  67 HSMDFVAYR A*03 CYC1  68 IADPFFRSAC*03: 04 BTN3A1  69 IEHPSMSVY B*18 TP53INP1  70 IESHPDNAL B*40NAE1 NEDD8  71 IEVEAVRF B*18 KIAA1217  72 IHILDVLVL B*15 CMTR1  73IIFDRPLLY A*03 DOLK  74 ILRDGITAGK A*03: 01 BTN3A1  75 ILWETVPSMA*02: 01 FNDC3B  76 IPAKPPVSF B*07: 02, TXNDC11 B*42: 01  77 IPAKPPVSFFB*07: 02 TXNDC11  78 IQAGIFQEF B*15 RAD1  79 IQILHQVL B*15 NPC1  80IVDRTTTVVNVEG class II SLC1A4  81 IVDRTTTVVNVEGDA class II SLC1A4  82KAMEAASSL A*02 WHSC1  83 KAVNPGRSL A*02 BFAR  84 KDARKGPLVP B*07 SETD8 85 KEENGVLVL B*40 PDIA4  86 KEFAAIVDV B*40 TXNDC11  87 KEGLILPETLB*40: 01 CREB3  88 KILKPVKKK A*03 CSNK2A1  89 KLGWLSSMTK A*03 COG1  90KLPLPLPPRL B*07 HSH2D  91 KLRELTQRY A*03 SPATC1L  92 KLSSLIILM A*02: 01SERPINH1  93 KPKDPLKISL B*07 PPP2R3C  94 KPQPRPQTL B*07 DYRK4  95KPRPPQGL B*07: 02, MOGS B*42: 01  96 KPRPPQGLVR B*07 MOGS  97 KPSTKALVLB*07 RAD1  98 KPYPNSEAARA B*55 CYC1  99 KQHGIPIPV B*27 ASS1 100KTEVHIRPK A*03: 01 LAP3 101 KTQLLPTSK A*33 ARHGAP11A 102 KVMLSALGML A*02CYC1 103 KYESIRLLF A*24 SNX14 14 104 KYPDSHLPTL A*24 KIAA1217 105LAALPGVSL A*02 LIME1 106 LADHTVHVL A*02: 01 ARHGAP11A 107 LAFPGEMLL A*02SLC1A4 108 LAHVGPRL A*02: 01 SLX1A 109 LEKEGLIL B*40 CREB3 110 LKIPISIEFB*15 MOGS 111 LLFPYILPPK A*02 SNX14 112 LLRFSQDNA A*02: 01 LAP3 113LPAEHGVL B*07 CREB3 114 LPKDVSPTQA B*55 COG1 115 LPPPPHVPL B*07: 02SLX1A 116 LPQLHSLVL B*07 LRRC47 117 LPVLLSYIGPSVNK class II NPC1 118LRFSQDNA C*07 LAP3 119 LYDVAGQGYL B*24: 02 PPP2R3C 120 MDLQPGNALKR n.a.LRRC47 121 MHGQPSPSL B*15 TMEM126B 122 mNIFRLTGDLSH class II KDELR2 123mPDDSYmVDYFKSISQ class II NPC1 124 mPDDSYmVDYFKSISQY class II NPC1 125MRLSLPLLL B*27 MZB1 126 MRLSLPLLLL B*27 MZB1 127 NEDFSFHY B*18P49770 EIF2B2 128 NEFPVFDEF B*18: 01 MB21D1 129 NEVIMTIGF B*18: 01P49770 EIF2B2 130 NGVLVLNDANFDNFV class II PDIA4 131 NIGQKEDFEEA A*02ASS1 132 NMDLMRADM A*02 LAP3 133 NPLLKHYRGPAGDA class II SLC1A5 134NPLLKHYRGPAGDAT class II SLC1A5 135 PELGPLPAL B*18, B*40 LRRC47 136PTENFSLPVL A*02 ZBTB21 137 PVLLSYIGPSVNK class II NPC1 138 QHYQQQQQVB*15: 10 BHLHA15 139 RAKDVIIPAK A*03 TXNDC11 140 RALDVDSGPL A*02 LIME1141 REEGTPLTL B*40: 01 NOC2L 142 RKDEDRKQF B*15 NOC2L 143 RKLAYRPPK B*15CYC1 144 RLGPPKRPPR A*30 MRPS12 145 RLKPFYLVPK A*03 MB21D1 146 RLQSKVTAKA*03 ASS1 147 RPFHGVVTSL B*07: 02 MOGS 148 RPGPPTRPL B*07: 02 FNDC3B 149FPHGGKSL B*07, TXNDC11 B*42: 01 150 RPKAQPTTL B*07; MED27 B*42: 01 151RPQLKGVVL B*07 MRPS12 152 RPRAPGPQ B*07 WFS1 153 RPRKAFLLLL B*07, PDIA4B*42: 01 154 RPRPPVLSV B*07 ZBTB21 155 RQFVVTRTKK A*03: 01 MRPL55 156RQYPEVIKY B*39 BAZ2B 157 RVAKTNSLR A*03: 01 Q53HL2 CDCA8 158 RVFPYSVFYA*03: 01 NPC1 159 RVNKVIIGTK A*03: 01 P49770 EIF2B2 160 RYFKGPELL A*24CSNK2A1 161 RYLDLFTSF A*24: 02 KDELR2 162 RYNPYLKR A*33 NBN 163RYSPVLSRF A*24: 02 COG1 164 RYSTQIHSF A*24: 02 BHLHA15 165 SEFDFFERLB*18: 01, SEMA4A B*40 166 SELVYTDVL B*40 MZB1 167 SESLPVRTL B*40: 01FNDC3B 168 SFDDAFKADS n.a. CMTR1 169 SFLDLARNIF A*24: 02 SLC1A5 170SHITRAFTV B*15 NPC1 171 SHSHVGYTL B*39 HSH2D 172 SHTPWIVII B*15 NAE1 173SIRRGFQVYK A*03 CYC1 174 SIYRGPSHTYK A*03 FNDC3B 175 SKDEARSSF B*15ARHGAP11A 176 SLGGKATTASQAKAV class II SERPINH1 177 SLLEQGLVEA A*02WHSC1 178 SMNVQGDYEPT A*02 ASS1 179 SPAHPKQTL B*07 BAZ2B 180 SPALKRLDLB*07: 02 COG1 181 SPALPGLKL B*07 TNFRSF13B 182 SPKSNDSDL B*42: 01 FNDC3B183 SPMPGTLTAL B*07 RAD1 184 SPPPPPPPP B*07 KIAA1217 185 SPQAETREA B*55NOC2L 186 SPRLSLLYL B*07 BFAR 187 SPRQALTDF B*07: 02 COG1 188 SPTKLPSIB*55 NBN 189 SPYLRPLTL B*07: 02 NUDT14 190 SRGDFVVEY C*07 SETD8 191SVYSPVKKK A*03 NUPL2 192 SYLNSVQRL A*24: 02 NUPL2 193 TASPLVKSV C*12ARHGAP11A 194 TEAQPQGHL B*40 BHLHA15 195 TEVIFKVAL B*18, B*40 TBC1D4 196TFLPFIHTI A*23: 01 BFAR 197 THAAEDIVYTL B*39: 01 FNDC3B 198 TKFGGIVVLB*15 NPC1 199 TLKSGDGITF B*15 NBN 200 TPAVGRLEV B*07, Q53HL2 B*42: 01201 TPEQQAAIL B*07 IRF9 202 TPSSRPASL B*07 UBL7 203 TRIGLAPVL B*15 CRLS1204 TVKATGPAL A*02 MRPL55 205 VAALAAHTTF A*24 TP53INP1 206 VDNIFILVQn.a. NPC1 207 VFDVLDGEEM A*24 CMTR1 208 VGGLSFLVNHDFS class II KDELR2209 VPAEGVRTA B*55 MOGS 210 VPLPPKGRVL B*42: 01 TMEM126B 211 VPLTRVSGGAAB*42: 01 SEMA4A 212 VPVGGLSFLVNHDF class II KDELR2 213 VPVGGLSFLVNHDFSclass II KDELR2 214 VPVGGLSFLVNHDFSP class II KDELR2 215VPVGGLSFLVNHDFSPL class II KDELR2 216 VPVGGLSFLVNHDFSPLE class II KDELR2217 VTDGKEVLL A*02 MOGS 218 YHAPPLSAITF B*15 ZBTB21 219 YILDPKQAL A*02TXNDC11 220 YLFAVNIKL A*02 CMTR1 221 YLYITKVLK A*03: 01 KDELR2 222YPDSKDLTM B*07 DYRK4 223 YPTIKILKKGQAVD class II PDIA4 224YPTIKILKKGQAVDY class II PDIA4 225 YPTIKILKKGQAVDYE class II PDIA4 226YPVFRILTL B*07 BTN3A1 227 YVFPGVTRL A*02 SPATC1L 228 YYLNEIQSF A*24SPATC1L

The abbreviations are as follows: TXNDC11=thioredoxin domain containing11, MOGS=mannosyl-oligosaccharide glucosidase, FNDC3B=fibronectin typeIII domain containing 3B, NUDT14=nudix (nucleoside diphosphate linkedmoiety X)-type motif 14, SLC1A5=solute carrier family 1 (neutral aminoacid transporter), member 5, ARHGAP11A=Rho GTPase activating protein11A, BHLHA15=basic helix-loop-helix family, member al 5, LRRC47=leucinerich repeat containing 47, PPP2R3C=protein phosphatase 2, regulatorysubunit B″, gamma, SLX1A=SLX1 structure-specific endonuclease subunithomolog A (S. cerevisiae), BAZ2B=bromodomain adjacent to zinc fingerdomain, 2B, NOC2L=nucleolar complex associated 2 homolog (S.cerevisiae), BTN3A1=butyrophilin, subfamily 3, member A1,TNFRSF13B=tumor necrosis factor receptor superfamily, member 13B,NPC1=Niemann-Pick disease, type C1, MRPS12=mitochondrial ribosomalprotein S12, NUPL2=nucleoporin like 2, CREB3=cAMP responsive elementbinding protein 3, TBC1 D4=TBC1 domain family, member 4, RAD1=RAD1checkpoint DNA exonuclease, NBN=nibrin, WFS1=Wolfram syndrome 1,WHSC1=Wolf-Hirschhorn syndrome candidate 1, ASS1=argininosuccinatesynthase 1, CYC1=cytochrome c-1, PDIA4=protein disulfide isomerasefamily A, member 4, LAP3=leucine aminopeptidase 3, KDELR2=KDEL(LysAsp-Glu-Leu) endoplasmic reticulum protein retention receptor,SLC1A4=solute carrier family 1 (glutamate/neutral amino acidtransporter), member 4, P49770=EIF2B2=eukaryotic translation initiationfactor 2B, subunit 2 beta, 39 kDa, SERPINH1=serpin peptidase inhibitor,Glade H (heat shock protein 47), member 1, DAP3=death associated protein3, IRF9=interferon regulatory factor 9, NAE1=NEDD8 activating enzyme E1subunit 1, Q53HL2=CDCA8=cell division cycle associated 8, KIAA1217,MED27=mediator complex subunit 27, MRPL55=mitochondrial ribosomalprotein L55, TMEM126B=transmembrane protein 126B, CMTR1=capmethyltransferase 1, MB21D1=Mab-21 domain containing 1, CSNK2A1=caseinkinase 2, alpha 1 polypeptide, COG1=component of oligomeric golgicomplex 1, MZB1=marginal zone B and B1 cell-specific protein,TP531NP1=tumor protein p53 inducible nuclear protein 1,HSH2D=hematopoietic SH2 domain containing, UBL7=ubiquitin-like 7,SPATC1L=spermatogenesis and centriole associated 1-like, SEMA4A=semadomain, immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4A, LIME1=Lck interacting transmembraneadaptor 1, SETD8=SET domain containing (lysine methyltransferase) 8,DYRK4=dual-specificity tyrosine-(Y)phosphorylation regulated kinase 4,BFAR=bifunctional apoptosis regulator, NDUFAF4=NADH dehydrogenase(ubiquinone) complex I, assembly factor 4, ZBTB21=zinc finger and BTBdomain containing 21, DOLK=dolichol kinase, SNX14=sorting nexin 14,NPC1=Niemann-Pick disease, type C1.

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, cancer, wherein said cancer is selectedfrom the group of lung cancer, brain cancer, hepatic cancer, kidneycancer, colorectal cancer, liver cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma,ovarian cancer, and esophageal cancer and other tumors that show anoverexpression of a protein from which a peptide SEQ ID No. 1 to SEQ IDNo. 228, and in particular myeloma.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of the SEQ ID NOs according to the following Table 2, andtheir uses in the immunotherapy of proliferative diseases, such as,cancer, wherein said cancer is selected from the group of lung cancer,brain cancer, hepatic cancer, kidney cancer, colorectal cancer, livercancer, pancreatic cancer, prostate cancer, leukemia, breast cancer,Merkel cell carcinoma, melanoma, ovarian cancer, and esophageal cancerand other tumors that show an overexpression of a protein from which apeptide SEQ ID No. 1 to SEQ ID No. 228, and in particular myeloma.

TABLE 2 Preferred peptides of the invention SEQ ID No. sequence HLAGene name   1 AASPVVAEY A*24 LIME1   2 AENAPSKEVL B*40 SLC1A5   4AFIQAGIFQEF A*23: 01 RAD1   5 AHSEQLQAL B*39 TXNDC11   6 AIILEAVNLPVDHclass II SLC1A5   7 AKRFDVSGY B*15 PDIA4  12 APKYGSYNVF B*42: 01 MOGS 15 APVGlmFLVAGKIV class II SLC1A5  16 APVGlmFLVAGKIVE class II SLC1A5 17 ASNPSNPRPSK A*30: 01 WHSC1 1  18 AVFIAQLSQQSLDF class II SLC1A5  19DALGAGILHHL A*02 SLC1A4  20 DEVLLQKL B*18 PPP2R3C  21DGDDVIIIGVFKGESDPAY class II PDIA4  22 DIKDTDVlmKR A*33 MB21D1  23DIQDPGVPR A*33 SEMA4A  24 DLFRYNPYLKR A*03 NBN  25 DLLDGFIAR A*03 CRLS1 26 DLNFPEIKR A*03 NOC2L  27 DLRPATDYHVR A*33 FNDC3B  28 DRYLLGTSL B*27ASS1  29 DSFERSNSL A*68: 02 TBC1D4  30 DTQSGSLLFIGR A*03 SERPINH1  32DVNNIGKYR A*03 LAP3  33 DVPDHIIAY A*03 KIAA1217  34 EGNPLLKHYRGPAGDAclass II SLC1A5  35 EGNPLLKHYRGPAGDAT class II SLC1A5  37 EIIEKNFDYLRA*03 TMEM126B  38 EITEVALEY A*26 TXNDC11  39 ENGVLVLNDANFDNFV class IIPDIA4  40 EQLYDLTLEY B*39 NOC2L  42 EYGHIPSF A*24: 02 ARHGAP11A  46FISGHTSEL A*02 MOGS  47 FKSPAASSF B*15 NUPL2  48 FLFQLLQLL A*02 SEMA4A 50 FNFLRNVSL B*08: 01 ARHGAP11A  52 GAKASTTSL C*03: 03 CMTR1  53GELIEVVHL B*40 NUDT14  54 GETAFAFHL B*40: 01 SLX1A  55 GEVAPSMFLB*40: 01 NPC1  56 GEVQDLLVRL B*40 BAZ2B  57 GKVQENSAY B*15 NOC2L  58GKYIFASIL B*15 SLC1A4  59 GNPLLKHYRGPAGDA class II SLC1A5  60 GPFSQFIKAB*55 FNDC3B  63 GYPTIKILKKGQAVDYE class II PDIA4  64 GYPTIKILKKGQAVDYEGclass II PDIA4  65 HPKQPEPSA B*42: 01 TXNDC11  66 HPKQPEPSAT B*42: 01TXNDC11  68 IADPFFRSA C*03: 04 BTN3A1  69 IEHPSMSVY B*18 TP53INP1  72IHILDVLVL B*15 CMTR1  79 IQILHQVL B*15 NPC1  80 IVDRTTTVVNVEG class IISLC1A4  81 IVDRTTTVVNVEGDA class II SLC1A4  84 KDARKGPLVP B*07 SETD8  85KEENGVLVL B*40 PDIA4  86 KEFAAIVDV B*40 TXNDC11  87 KEGLILPETL B*40: 01CREB3  89 KLGWLSSMTK A*03 COG1  94 KPQPRPQTL B*07 DYRK4  96 KPRPPQGLVRB*07 MOGS  98 KPYPNSEAARA B*55 CYC1  99 KQHGIPIPV B*27 ASS1 101KTQLLPTSK A*33 ARHGAP11A 102 KVMLSALGML A*02 CYC1 104 KYPDSHLPTL A*24KIAA1217 106 LADHTVHVL A*02: 01 ARHGAP11A 107 LAFPGEMLL A*02 SLC1A4 108LAHVGPRL A*02: 01 SLX1A 109 LEKEGLIL B*40 CREB3 110 LKIPISIEF B*15 MOGS112 LLRFSQDNA A*02: 01 LAP3 113 LPAEHGVL B*07 CREB3 114 LPKDVSPTQA B*55COG1 117 LPVLLSYIGPSVNK class II NPC1 118 LRFSQDNA C*07 LAP3 119LYDVAGQGYL B*24: 02 PPP2R3C 120 MDLQPGNALKR n.a. LRRC47 122 mNIFRLTGDLSHclass II KDELR2 123 mPDDSYmVDYFKSISQ class II NPC1 124 mPDDSYmVDYFKSISQYclass II NPC1 126 MRLSLPLLLL B*27 MZB1 127 NEDFSFHY B*18 P49770 EIF2B2128 NEFPVFDEF B*18: 01 MB21D1 130 NGVLVLNDANFDNFV class II PDIA4 131NIGQKEDFEEA A*02 ASS1 132 NMDLMRADM A*02 LAP3 133 NPLLKHYRGPAGDAclass II SLC1A5 134 NPLLKHYRGPAGDAT class II SLC1A5 136 PTENFSLPVL A*02ZBTB21 137 PVLLSYIGPSVNK class II NPC1 138 QHYQQQQQV B*15: 10 BHLHA15139 RAKDVIIPAK A*03 TXNDC11 140 RALDVDSGPL A*02 LIME1 142 RKDEDRKQF B*15NOC2L 143 RKLAYRPPK B*15 CYC1 145 RLKPFYLVPK A*03 MB21D1 148 RPGPPTRPLB*07: 02 FNDC3B 149 RPHGGKSL B*07, TXNDC11 B*42: 01 152 RPRAPGPQ B*07WFS1 158 RVFPYSVFY A*03: 01 NPC1 161 RYLDLFTSF A*24: 02 KDELR2 162RYNPYLKR A*33 NBN 166 SELVYTDVL B*40 MZB1 168 SFDDAFKADS n.a. CMTR1 169SFLDLARNIF A*24: 02 SLC1A5 170 SHITRAFTV B*15 NPC1 172 SHTPWIVII B*15NAE1 175 SKDEARSSF B*15 ARHGAP11A 176 SLGGKATTASQAKAV class II SERPINH1178 SMNVQGDYEPT A*02 ASS1 185 SPQAETREA B*55 NOC2L 188 SPTKLPSI B*55 NBN193 TASPLVKSV C*12 ARHGAP11A 194 TEAQPQGHL B*40 BHLHA15 196 TFLPFIHTIA*23: 01 BFAR 197 THAAEDIVYTL B*39: 01 FNDC3B 198 TKFGGIVVL B*15 NPC1203 TRIGLAPVL B*15 CRLS1 204 TVKATGPAL A*02 MRPL55 205 VAALAAHTTF A*24TP53INP1 206 VDNIFILVQ n.a. NPC1 207 VFDVLDGEEM A*24 CMTR1 208VGGLSFLVNHDFS class II KDELR2 209 VPAEGVRTA B*55 MOGS 210 VPLPPKGRVLB*42: 01 TMEM126B 211 VPLTRVSGGAA B*42: 01 SEMA4A 212 VPVGGLSFLVNHDFclass II KDELR2 213 VPVGGLSFLVNHDFS class II KDELR2 214 VPVGGLSFLVNHDFSPclass II KDELR2 215 VPVGGLSFLVNHDFSPL class II KDELR2 216VPVGGLSFLVNHDFSPLE class II KDELR2 218 YHAPPLSAITF B*15 ZBTB21 219YILDPKQAL A*02 TXNDC11 222 YPDSKDLTM B*07 DYRK4 223 YPTIKILKKGQAVDclass II PDIA4 224 YPTIKILKKGQAVDY class II PDIA4 225 YPTIKILKKGQAVDYEclass II PDIA4

Many of the peptides according to the present invention are also foundon other tumor types and can, thus, also be used in the immunotherapy ofother indications.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofcancer, wherein said cancer is selected from the group of lung cancer,brain cancer, hepatic cancer, kidney cancer, colorectal cancer, livercancer, pancreatic cancer, prostate cancer, leukemia, breast cancer,Merkel cell carcinoma, melanoma, ovarian cancer, and esophageal cancerand other tumors that show an overexpression of a protein from which apeptide SEQ ID No. 1 to SEQ ID No. 228, and in particular myeloma.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II, or in anelongated form, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 228.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 228, preferably containing atleast one SEQ ID No. according to table 2, or a variant amino acidsequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are lung cancer, brain cancer,hepatic cancer, kidney cancer, colorectal cancer, liver cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, and esophageal cancer cells, andpreferably myeloma cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably myeloma. Themarker can be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying expression products (polypeptides) of the peptides accordingto the invention.

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or even longer, andin case of MHC class II peptides (e.g. elongated variants of thepeptides of the invention) they can be as long as 15, 16, 17, 18, 19, 20or 23 or more amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 2 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 −(1-Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles mightbe enriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype from allele AllelePopulation frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein preferably bind to HLA-A*02. Avaccine may also include pan-binding MHC class II peptides. Therefore,the vaccine of the invention can be used to treat cancer in patientsthat are A*02 positive, whereas no selection for MHC class II allotypesis necessary due to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 228 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 228, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 228. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 228, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 4.

TABLE 4 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 or 23 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 228.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 228 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, Gen Bank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and the references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as recrystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from myeloma samples with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue obtained from myeloma patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from myeloma samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary myeloma samples confirmingtheir presentation on myeloma.

TUMAPs identified on multiple myeloma and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably myeloma that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on human myelomasamples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy plasma cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes. Moreover, the peptidesthemselves are strongly over-presented on tumor tissue—“tumor tissue” inrelation to this invention shall mean a sample from a patient sufferingfrom myeloma, but not on normal tissues.

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. myeloma cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention. Furthermore, the peptides whencomplexed with the respective MHC can be used for the production ofantibodies and/or TCRs, in particular sTCRs, according to the presentinvention, as well. Respective methods are well known to the person ofskill, and can be found in the respective literature as well. Thus, thepeptides of the present invention are useful for generating an immuneresponse in a patient by which tumor cells can be destroyed. An immuneresponse in a patient can be induced by direct administration of thedescribed peptides or suitable precursor substances (e.g. elongatedpeptides, proteins, or nucleic acids encoding these peptides) to thepatient, ideally in combination with an agent enhancing theimmunogenicity (i.e. an adjuvant). The immune response originating fromsuch a therapeutic vaccination can be expected to be highly specificagainst tumor cells because the target peptides of the present inventionare not presented on normal tissues in comparable copy numbers,preventing the risk of undesired autoimmune reactions against normalcells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are HAVCR1-001 peptides capable of binding to TCRs andantibodies when presented by an MHC molecule. The present descriptionalso relates to nucleic acids, vectors and host cells for expressingTCRs and peptides of the present description; and methods of using thesame.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an HAVCR1-001peptide-HLA molecule complex with a binding affinity (KD) of about 100μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM orless. More preferred are high affinity TCRs having binding affinities ofabout 1 μM or less, about 100 nM or less, about 50 nM or less, about 25nM or less. Non-limiting examples of preferred binding affinity rangesfor TCRs of the present invention include about 1 nM to about 10 nM;about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM toabout 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM;about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM toabout 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for an HAVCR1-001 peptide-HLAmolecule complex of 100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a HAVCR1-001 peptide-HLA molecule complex, whichis at least double that of a TCR comprising the unmutated TCR alphachain and/or unmutated TCR beta chain. Affinity-enhancement oftumor-specific TCRs, and its exploitation, relies on the existence of awindow for optimal TCR affinities. The existence of such a window isbased on observations that TCRs specific for HLA-A2-restricted pathogenshave KD values that are generally about 10-fold lower when compared toTCRs specific for HLA-A2-restricted tumor-associated self-antigens. Itis now known, although tumor antigens have the potential to beimmunogenic, because tumors arise from the individual's own cells onlymutated proteins or proteins with altered translational processing willbe seen as foreign by the immune system. Antigens that are upregulatedor overexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to HAVCR1-001 can be enhanced by methods well known in theart.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/HAVCR1-001 monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with HAVCR1-001, incubating PBMCs obtained from the transgenicmice with tetramer-phycoerythrin (PE), and isolating the high avidityT-cells by fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus post-transcriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of overcoming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutics such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 228, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, 1030, 1031, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, betaglucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomateous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualisation of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 228according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 228, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 228 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:228 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 228, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 228.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of myeloma.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 228 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are myeloma cells or other solid orhematological tumor cells.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of myeloma. The present invention also relates to the use ofthese novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a myeloma marker (poly)peptide,delivery of a toxin to a myeloma cell expressing a cancer marker gene atan increased level, and/or inhibiting the activity of a myeloma markerpolypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length myeloma marker polypeptides or fragments thereofmay be used to generate the antibodies of the invention. A polypeptideto be used for generating an antibody of the invention may be partiallyor fully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 228polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the myeloma marker polypeptide usedto generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed lung cancers or frozentissue sections. After their initial in vitro characterization,antibodies intended for therapeutic or in vivo diagnostic use are testedaccording to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating myeloma, theefficacy of the therapeutic antibody can be assessed in various wayswell known to the skilled practitioner. For instance, the size, number,and/or distribution of lung cancer in a subject receiving treatment maybe monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment oflung cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 228, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) who describe the development of cowpeamosaic virus as a high-yielding system for the presentation of foreignpeptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 228.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from myeloma, themedicament of the invention is preferably used to treat myeloma.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in myeloma cells of patientswith various HLA-A HLA-B and HLA-C alleles. It may contain MHC class Iand MHC class II peptides or elongated MHC class I peptides. In additionto the tumor associated peptides collected from several myelomas, thewarehouse may contain HLA-A*02 and HLA-A*24 marker peptides. Thesepeptides allow comparison of the magnitude of T-cell immunity induced byTUMAPS in a quantitative manner and hence allow important conclusion tobe drawn on the capacity of the vaccine to elicit anti-tumor responses.Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And thirdly, it may allow conclusions to be drawn, regardingthe status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, myeloma samples from patientsand blood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the myeloma compared with arange of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as frommyeloma patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (al) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumine). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from myeloma cells and since it was determined that thesepeptides are not or at lower levels present in normal tissues, thesepeptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for myeloma. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

As loss or down-regulation of HLA expression on target cells mightseverely hamper the effectiveness of T-cell based immunotherapy, theinventors quantified HLA class I and II surface molecule counts onprimary myeloma cells compared to autologous hematopoietic cells andplasma cells derived from the bone marrow of HVs. In MM patients (n=20)HLA class I expression was found to be heterogeneous with meanexpression levels on CD38⁺CD138⁺ myeloma cells of 416,000±54,500, whichwas found to be significantly higher as compared to autologous normalCD19⁺CD20⁺ B cells (198,5000±20,500, P=0.001), CD3⁺ T cells(167,500±15,500, P=0.0002) and CD34⁺CD38⁻ HPCs (204,000±32,500, P=0.002,FIG. 1A). In addition, HLA class I expression on primary MM cells wasalso found to be significantly higher than that on CD38⁺CD138⁺ plasmacells of HVs (n=15, 291,500±25,500, P<0.05; FIG. 10). No significantdifferences in HLA class I expression were observed when comparingnormal B cells, T cells and HPCs of MM patients to the correspondingcell populations of HVs (supplemental FIG. 1B). HLA-DR expression levelson myeloma cells were generally found to be much lower than HLA class Ilevels. Mean HLA-DR surface molecule counts on myeloma cells(27,000±7,000) showed no significant difference compared to autologousHPCs (35,000±5,000) and T cells (18,000±13,000) or plasma cells of HVs(39,500±5,000) (FIGS. 1B and 1D). HLA-DR expression of MM patientCD19⁺CD20⁺ B cells (104,000±7,000) was significantly higher compared tomyeloma cells (P<0.0001). No correlation of HLA surface expression onmyeloma cells with patient characteristics such as sex, age, diseasestage, risk classification or prior therapy was observed.

Mapping the HLA class I ligandomes of 10 myeloma patients and 5 MCLs,the inventors identified a total of 17,583 different peptidesrepresenting 7,574 source proteins, attaining >80% of the maximumattainable coverage (FIG. 2A). The mean number of unique peptideidentifications was 1,059 IDs for primary myeloma samples and 2,243 IDsfor MCLs. Overall, peptides restricted by 20 different HLA-A and -Ballotypes were identified in this study, covering 99.3% of the Caucasianpopulation (calculated according to [52]).

As controls, the inventors analyzed the HLA class I ligandomes of 45 HVderived samples (30 PBMC, 10 BMNC and 5 granulocyte specimens)identifying a total of 20,171 different peptides representing 7,729source proteins. The HLA allotype distribution in the HV cohortcovered >80% of HLA-A and -B alleles in the MM sample cohort [53].Analysis of HLA class II ligandomes was performed for 7 MM patients and5 MCLs. A total of 6,076 unique peptides representing 1,743 sourceproteins were identified. The HLA class II HV cohort (13 PBMC, 5 BMNC, 5granulocyte specimens) yielded 2,899 different peptides representing 889source proteins.

In order to identify myeloma-associated antigens the inventorscomparatively analyzed the HLA ligandomes of the MM sample and HVcohorts at the source protein level. Overlap analysis of HLA ligandsource proteins identified 2,412 proteins (corresponding to 31.8% of themapped MM HLA source proteome) to be exclusively represented in the HLAligandomes of MM samples. Of these MM-exclusive source proteins, 68.3%were solely identified on MCL samples, whereas 13.2% of proteins werefound to be presented both, on MCLs and primary MM samples. A fractionof 18.5% of myeloma-exclusive source proteins was found to be restrictedto primary MM samples (FIG. 2B). In order to identify broadly presentedtumor-associated antigens, myeloma-exclusive source proteins were rankedaccording to their frequencies of representation in the MM sample cohort(FIG. 2C). To statistically assess and optimize the stringency ofantigen identification, the inventors simulated randomized virtualligandomes in silico and calculated the resultant number offalse-positive TAAs at different frequencies of representation (FIG.2D). The inventors set the frequency threshold for HLA class I tumorassociated antigen (“TAA”) definition to >25% of myeloma-exclusiveantigen presentation, yielding 58 TAAs with an estimated false discoveryrate (FDR) of 4.1%. This novel panel of frequently presentedmyeloma-associated antigens was represented by 197 unique HLA class Iligands and constitutes 0.8% of the mapped myeloma HLA ligand sourceproteome. KEGG pathway analysis [54] and functional annotationclustering of these antigens with respect to their biological function(GO Term BP FAT, [55]) did not identify any statistically significantoverrepresented pathways or functional clusters. Notably, theproto-oncogene MMSET was identified as a TAA showing representation in33% of MM patient ligandomes and was found to be represented by 3different HLA ligands (ASNPSNPRPSK (HLA-A*30:01) (SEQ ID NO. 17),KAMEAASSL (A*02:01) (SEQ ID NO. 82), SLLEQGLVEA (A*02:01) (SEQ ID NO.177)). Moreover, MMSET was detected on both of the two MM patients withthe oncogenic translocation t(4;14), but only on 1/6 (17%) patientswithout this aberration.

Representation of Established Myeloma-Associated Antigens in the HLAClass I Ligandome

Based on the inventors' extensive HLA ligandome dataset, the inventorsinvestigated the presentation of established myeloma-associated antigenswithin the different sample cohorts. The inventors identified 73different HLA ligands representing 22/25 (88%) of previously describedmyeloma antigens [42]. The inventors found 9 of the 22 detectableantigens (41%) to be exclusively presented on MM samples, 10/22 (45.5%)antigens to be represented both on MM and HV samples, and 3/22 (13.6%)exclusively presented on HV derived samples (FIG. 3A). Of note, 7/9(77.8%) MM-exclusive antigens were only detectable on MCLs. Only for 2/9(22.2%) of these MM-exclusive antigens HLA ligands were detected onprimary MM patient samples (FIG. 3B). For reference, only 7/58 (12.1%)of the newly defined myeloma antigens showed presentation exclusively onMCLs, whereas the majority of 51/58 (87.9%) of antigens was alsopresented on primary MM patient samples as well, which underlines theirpotential as clinical target antigens (FIG. 3C).

Moreover, unsupervised clustering of source protein presentation in theHLA ligandomes revealed the cluster of MCLs to be highly distinct fromprimary MM samples (supplemental FIGS. 2A-2D).

Analysis of HLA Class II Ligandomes Identifies Potentially SynergisticVaccine Candidates

As the direct involvement of CD4⁺ T cells in tumor control isestablished [56], the inventors further aimed to identify HLA class IIantigens. Overlap analysis of HLA class II ligand source proteinsidentified 1,135 myeloma exclusive antigens (FIG. 4A). Comparativeprofiling of HLA class II ligandomes identified a single antigen (TFRC)represented by 67 HLA class II ligands showing MM-exclusive presentationat FDR <5% (FIGS. 4B, 4C). Functional characterization of the mostabundant TFRC peptide (NSVIIVDKNGRLV) (SEQ ID NO. 237). by IFNγ ELISPOTrevealed memory T-cell responses in 2/5 MM patients.

As CD4⁺ T cells play pivotal roles in the induction and maintenance ofantigen-specific CD8⁺ T-cell responses [57-59], the inventorsimplemented a second approach to identify potentially synergistic HLAclass II restricted peptides derived from HLA class I TAAs. Overlapanalysis of the 58 HLA class I antigens with the 1,135 HLA class IIpresented MM exclusive proteins identified a panel of 6 class-spanningantigens, including APVGIMFLVAGKIVE (SEO ID NO: 16), MPDDSYMVDYFKSISO(SEO ID NO: 123), GYPTIKILKKGOA VDYEG (SEO ID NO: 64), VPVGGLSFLVNHDFSPL(SEO ID NO: 215), and IVDRTTTWNVEG (SEO ID NO: 80), represented by 31peptides (FIGS. 4D, 4E; supplemental Table 5). Functionalcharacterization of synergistic HLA class II ligands revealedpeptide-specific T-cell responses in myeloma patients for 3/5 testedpeptides (FIG. 4E).

The overall comparison of the HLA class I and II ligandomes of MMsamples revealed 80% (1,622) of HLA class II presented proteins to bealso presented on HLA class I (FIG. 4F). Functional annotationclustering (GO Term CC clustering using DAVID [55]) was performed on thetop 500 most frequently presented proteins in each HLA class to identifythe cellular compartments from which these proteins derive. Antigenspresented on class I displayed highly enriched clusters for nuclearproteins as well as for ribosomal, cytoskeletal and vesicle-derivedproteins. Notably, this pattern was recapitulated in the clustering ofproteins presented on both HLA classes, albeit with a higher ranking andan almost 3-fold higher enrichment for vesicle-derived proteins.

HLA Class II Presented Antigens Showed Intermediate Enrichment forPlasma Membrane, Vesicle-Derived and Lysosomal Proteins (SupplementalTable 6).

HLA class I TAAs are targeted by spontaneous T-cell responses in myelomapatients Functional characterization of the novel myeloma antigens wasperformed in panels of 11 HLA-A*02 and 2 HLA-B*07 restricted peptides,including 2 HLA-A*02 ligands derived from MMSET, e.g., KAMEAASSL (SEO IDNO: 82) and SLLEOGL VEA (SEO ID NO: 177), one HLA-A*02 ligand derivedfrom SLC 1A5, e.g., FVFPGELLL (SEO ID NO: 51), one HLA-A*02, ligandderived from SEMA4A, e.g., FLFOLLOLL (SEO ID NO: 48), one HLA-B*07ligand derived from SLXIA, e.g., LPPPPHVPL (SEO ID NO: 115), and oneHLA-A*02 ligand derived from SLXIA, e.g., LAHVGPRL (SEO ID NO: 108)(FIG. 5A). Myeloma-associated peptides were evaluated in 12-day recallIFNγ ELISPOT assays using PBMC obtained from MM patients and HVs. Theinventors observed IFNγ secretion for 5/11 A*02 ligands and 1/2 B*07ligands in myeloma patients, as shown exemplarily in FIG. 5C. Bothpeptides (P₁ and F₂) derived from MMSET showed specific T-cellrecognition in 2/16 (13%) and 1/8 (13%) of MM patients, respectively.Importantly, no myeloma peptide-specific IFNγ secretion was observed in10 HLA-matched healthy controls (FIG. 5B). Notably, T-cell responseswere only observed for myeloma-associated peptides identified on primarymyeloma samples (10/13), and never for the 3/13 peptides identified onMCLs only. The frequencies of peptide-specific T-cell responses detectedin MM patients by ELISPOT were generally in the same range as thefrequencies of presentation of the respective peptide inallotype-matched ligandomes of MM patients (FIG. 5A). Due to limitationsin the numbers of cells available for analysis, further controls withtarget cells expressing the corresponding antigens could not beperformed. The inventors therefore cannot exclude that T-cell reactivityis directed against impurities contained in the synthetic peptide batch.Indeed, it is well known that synthetic peptides contain impurities,e.g. peptides modified with a protecting group, and that theseimpurities are immunogenic. However, HLA-A*02 and -B*07 restrictedcontrol peptides derived from benign tissues (HV exclusive HLA ligands)used in all ELISPOTs in the study at hand did never result insignificant IFNγ release (FIG. 5C).

Antigen-Specific T Cells can be Induced In Vitro from Naïve T Cells ofMM Patients or HVs

To assess whether myeloma antigen-specific T-cell responses can beinduced from naïve T cells in vitro the inventors isolated CD8⁺ T cellsfrom one healthy individual and one MM patient. The inventors performedaAPC-primings using the MMSET-derived peptide SLLEQGLVEA (SEQ ID NO.177) (P₂). Using HV-derived CD8⁺ T cells, a population of 0.403%P₂-tetramer positive CD8⁺ T cells was detected after in vitro priming.No tetramer-positive T-cell populations >0.1% were detectable ex vivo.After priming of T cells from an MM patient without previous T-cellreactivity for P2 (as detected by 12d-recall IFNγ-ELISPOT and ex vivotetramer staining), the inventors detected the induction of a smallpopulation of 0.236% P₂-tetramer positive CD8⁺ T cells (FIG. 5E).Importantly, control stainings performed with an A*02-tetramercontaining a non-relevant A*02 control peptide were performed inparallel on cells derived from the same wells as used for the relevantstaining and did not yield any specific tetramer-positive T-cellpopulations (FIG. 5D).

Quantification of HLA surface expression on different cell populationsin the bone marrow of myeloma patients and healthy volunteersdemonstrated that HLA-loss or down-regulation on malignant plasma cellsis of no concern, even in patients who received prior therapy.Comparative analysis of the HLA ligandomes of these cell populationsrevealed distinct antigenic signatures and identified a panel ofmyeloma-associated antigens.

Importantly, a substantial proportion of established myeloma-antigenswere found to be only infrequently presented on primary myelomas or toshow suboptimal degrees of myeloma-specificity. Of note, the majority ofthese antigens were selectively detected on myeloma cell lines but notin primary samples, indicating that selection of pathophysiologicallyrelevant antigens should be based on analysis of primary tumor samples.

A notable exception was the established myeloma-associated proteinMMSET, which is currently being investigated as a target for the therapyof poor-prognosis t(4;14) myeloma patients [73-76]. AlthoughMMSET-derived peptides were frequently identified on t(4;14) myelomasamples, the inventors also detected MMSET peptides in the HLAligandomes of a t(4;14)-negative patient and one t(4;14)-negative MCL(U266). Strikingly, functional characterization by ELISPOT revealedmemory T-cell responses targeting these MMSET-derived epitopesexclusively in myeloma patients and not in HV. This suggestsmyeloma-dependent priming of anti-MMSET T-cell responses in vivo in MMpatients, which underscores the pathophysiological relevance of thisantigen. In concordance with the HLA ligandomics data, the inventorsfound these T-cell responses not to be restricted to t(4;14) myelomapatients. Results of in vitro primings suggest that MMSET-specific CD8⁺T-cell responses can be induced from naïve T cells, both in healthyindividuals and, importantly, also in myeloma patients, albeit withlimited magnitudes. With the current strategies focusing on inhibitionof MMSET by small molecules or siRNAs [77, 78], the inventors'identification of myeloma-exclusive MMSET-derived T-cell epitopesprovides new options for targeting MMSET by T-cell based immunotherapy.Notably, this therapeutic strategy may not necessarily have to berestricted to t(4;14) myelomas, as the inventors observedMMSET-presentation and immune recognition irrespective of the mutationalstatus. This might be explained by the distorted correlation of geneexpression and HLA restricted antigen presentation as well as by thesubclonal distribution of t(4;14) in myeloma cells and genomicplasticity occurring over the course of disease [72, 79].

Together, the inventors' findings illustrate how antigen identificationguided by HLA ligandomics can pinpoint novel MM-associated T-cellepitopes and allows to directly assessing antigen distribution patternsin patient cohorts. In parallel to the inventors' findings with MMSET,the inventors' study features an extensive panel of novel antigenspreviously not associated with myeloma or cancer in general. Analogouslyto MMSET, the inventors detected pre-existing T cell responses against asubstantial proportion of these targets in myeloma patients, indicatinga high enrichment for relevant MM-associated antigens. In conclusion,the inventors' ligandome-centric study may guide the design of futureantigen-specific T-cell immunotherapy in multiple myeloma.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A to 1D show the HLA class I & II surface expression on myelomapatient and HV bone marrow cells. Quantification of HLA surfaceexpression was performed using a bead-based flow cytometric assay. (FIG.1A) HLA class I and (FIG. 1B) HLA-DR expression on CD38⁺CD138⁺ primarymyeloma cells compared to autologous CD34⁺CD38⁻ hematopoietic progenitorcells, CD19⁺CD20⁺ B cell and CD3⁺ T cells. (FIG. 10) HLA class I and(FIG. 1D) HLA-DR expression on primary MM cells compared to bonemarrow-derived plasma cells of HVs. Abbreviations: MM, multiple myeloma;HV, healthy volunteers; n.s., not significant; *P<0.05, **P<0.01,***P<0.001

FIGS. 2A to 2D show the comparative HLA ligandome profiling andidentification of myeloma associated antigens. (FIG. 2A) Saturationanalysis of HLA class I ligand source protein identifications in MMpatients. Number of unique HLA ligand source protein identifications asa function of cumulative HLA ligand source protein identifications in 10MM patients. Exponential regression allowed for the robust calculation(R²=0,99) of the maximum attainable number of different source proteinidentifications (dashed line). The dotted line depicts the sourceproteome coverage achieved in the inventors' MM patient cohort. (FIG.2B) Overlap analysis of HLA class I ligand source proteins of primary MMsamples (n=10), MCLs (n=5) and HV samples (total n=45: PBMC (n=30), BMNC(n=10), granulocytes (n=5)). (FIG. 2C) Comparative profiling of HLAligand source proteins based on the frequency of HLA restrictedpresentation in MM and HV ligandomes. Frequencies of MMs/HVs positivefor HLA restricted presentation of the respective source protein(x-axis) are indicated on the y-axis. The box on the left highlights thesubset of myeloma-associated antigens showing MM-exclusive presentationin >25% of myeloma samples. (FIG. 2D) Statistical assessment offalse-positive myeloma-antigen identifications at different thresholdvalues. The numbers of original TAAs identified based on the analysis ofthe MM and HV cohorts were compared with random virtual TAAs. Virtual MMand HV samples were generated in silico based on random weightedsampling from the entirety of protein identifications in both originalcohorts. These randomized virtual ligandomes of defined size (n=957proteins, which is the mean number of protein identifications in allanalyzed samples) were used to define TAAs based on simulated cohorts of15 MM versus 45 HV samples. The process of protein randomization, cohortassembly and TAA identification was repeated 1,000 times and the meanvalue of resultant virtual TAAs was calculated and plotted for thedifferent threshold values. The corresponding false discovery rates forany chosen TAA threshold are listed below the x axis. Abbreviations: ID,identifications; MM, multiple myeloma; MCL, myeloma cell line; HV,healthy volunteer; PBMC, peripheral mononuclear blood cell; BMNC, bonemarrow mononuclear cell; TAA, tumor-associated antigen; sum, summary;FDR, false discovery rate.

FIGS. 3A to 3C show the representation of established myeloma-associatedantigens in the HLA ligandomes of MM and HV. (FIG. 3A) Representation ofpreviously described MM-associated antigens in HLA class I ligandomes.Bars indicate relative representation [%] of respective antigens by HLAclass I ligands on primary MM samples, MCLs and HV samples. Dashed linesdivide the antigens into 4 groups according to their degree ofMM-association (MM & MCL-exclusive, MCL-exclusive, mixed presentation,HV-exclusive). (FIGS. 3B, 3C) Distribution of myeloma-exclusive antigenpresentation for (FIG. 3B) previously described antigens and (FIG. 3C)ligandome-defined tumor-associated antigens on MCLs (white) and MM+MCLs(shaded). Abbreviations: MM, multiple myeloma; MCL, myeloma cell line;HV, healthy volunteer.

FIGS. 4A to 4F shows the identification of synergistic HLA class IIrestricted myeloma-associated antigens. (FIG. 4A) Overlap analysis ofHLA class II ligand source proteins of primary MM samples (n=7), MCLs(n=5) and HV samples (total n=23: PBMC (n=13), BMNC (n=5), granulocytes(n=5)). (FIG. 4B) Statistical analysis of false-positive myeloma-antigenidentifications at different threshold values, as described in FIG. 2.Randomized virtual ligandome sizes were set to 226 proteins and TAAswere defined based on simulated cohorts of 12 MM versus 23 HV samples.(FIG. 4C) Comparative profiling of HLA class II ligand source proteinsbased on the frequency of HLA restricted presentation in MM and HVligandomes. Frequencies of MMs/HVs positive for HLA restrictedpresentation of the respective source protein (x-axis) are indicated onthe y-axis. (FIG. 4D) Overlap analysis of HLA class I TAAs (n=58) andHLA class II MM-exclusive antigens (n=1135). (FIG. 4E) HLA class I TAAs,which also yield potentially synergistic HLA class II ligands. (FIG. 4F)Overlap analysis comprising the entire HLA class I and II ligand sourceproteomes of MM samples. Abbreviations: MM, multiple myeloma; MCL,myeloma cell line; HV, healthy volunteer; PBMC, peripheral mononuclearblood cell; BMNC, bone marrow mononuclear cell; TAA, tumor-associatedantigen; sum, summary; FDR, false discovery rate; rep., representation.

FIGS. 5A to 5E show the functional characterization ofmyeloma-associated antigens. (FIG. 5A) Myeloma-associated T cellepitopes with their corresponding HLA restrictions and frequencies ofimmune recognition by myeloma patient derived T cells in IFNγ-ELISPOTassays. (FIG. 5B) Example of myeloma-associated T cell epitopesevaluated in an IFNγ-ELISPOT using HV PBMC. An EBV epitope mixcontaining the frequently recognized peptides BRLF109-117 YVLDHLIW(A*02) (SEQ ID NO. 229) and EBNA3247-255 RPPIFIRRL (SEQ ID NO. 230)(B*07 served as positive control. Benign-tissue derived peptidesKLFEKVKEV (SEQ ID NO. 231) (HLA-A*02) and KPSEKIQVL (B*07) (SEQ ID NO.232) served as negative control. (FIG. 5C) Examples ofmyeloma-associated T cell epitopes evaluated in IFNγ-ELISPOTs using MMpatient PBMC (n=3). Results are shown only for immunoreactive peptides.An EBV epitope mix containing five frequently recognized peptides[BRLF109-117 YVLDHLIW (A*02) (SEQ ID NO. 229), EBNA3471-479 RLRAEAQVK(A*03) (SEQ ID NO. 233), EBNA3247-255 RPPIFIRRL (B*07) (SEQ ID NO. 230),BZLF1190-197 RAKFKQLL (B*08) (SEQ ID NO. 234), EBNA6162-171 AEGGVGWRHW(B*44) (SEQ ID NO. 235)] was used as positive control. Benign-tissuederived peptides KLFEKVKEV (SEQ ID NO. 231) (HLA-A*02) and KPSEKIQVL(B*07) (SEQ ID NO. 232) served as negative control. (FIGS. 5D, 5E)Tetramer staining of CD8⁺ T cells after 3 cycles of aAPC-based in vitroprimings using T cells derived from (FIG. 5D) a healthy individual and(FIG. 5E) a myeloma patient: 1^(st) column: P₂-tetramer staining of CD8⁺T cells primed with P₂-aAPCs (SLLEQGLVEA, A*02 (SEQ ID NO. 177)); 2^(nd)column: ex vivo P₂-tetramer staining of CD8⁺ T cells; 3^(rd) column:control staining with A*02-tetramer containing a non-relevant A*02restricted control peptide (KAMEAASSL, A*02 (SEQ ID NO. 82)) on CD8⁺ Tcells derived from the same population as T cells depicted in the 1^(st)column. 4^(th) column: positive control: tetramer staining of CD8⁺ Tcells primed with CMV-aAPCs (NLVPMVATV, A*02 (SEQ ID NO. 236)).Abbreviations: MM, multiple myeloma; UPN, uniform patient number; neg.,negative; pos., positive.

FIGS. 6A and 6B show presentation of peptides SEQ ID NO: 107 and 177 ontissues other than myeloma. FIG. 6A) Normal tissues tested negative forthe peptide were: 6 adipose tissues, 8 adrenal glands, 24 blood cellsamples, 15 blood vessels, 10 bone marrows, 13 brains, 7 breasts, 9esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 25 largeintestines, 24 livers, 49 lungs, 7 lymph nodes, 12 nerves, 3 ovaries, 13pancreases, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7placentas, 1 pleura, 4 prostates, 7 salivary glands, 9 skeletal muscles,11 skins, 9 small intestines, 11 spleens, 8 stomachs, 5 testes, 3 thymi,5 thyroid glands, 16 tracheas, 7 ureters, 8 urinary bladders, 6 uteri.In addition to MM, the peptide was found presented on: 1 cell line(melanoma), 1 normal tissue (spleen), 5 cancer samples (AML, 2gallbladder cancers, 1 hepatocellular carcinoma, 1 melanoma). FIG. 6B)Normal tissues tested negative for the peptide were: 6 adipose tissues,8 adrenal glands, 24 blood cell samples, 15 blood vessels, 10 bonemarrows, 9 brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16hearts, 17 kidneys, 23 large intestines, 24 livers, 49 lungs, 7 lymphnodes, 10 nerves, 3 ovaries, 13 pancreases, 6 parathyroid glands, 1peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7salivary glands, 9 skeletal muscles, 11 skins, 8 small intestines, 11spleens, 8 stomachs, 5 testes, 3 thymi, 5 thyroid glands, 15 tracheas, 7ureters, 8 urinary bladders, 6 uteri. In addition to MM, the peptide wasfound presented on: 6 cell-lines (5 leukemias, 1 kidney cancer), 4brains, 1 central nerve, 2 colons, 1 peripheral nerve, 1 prostate, 1small intestine, 1 spleen, 1 trachea, 1 bile duct cancer, 12 braincancers, 2 breast cancers, 3 colon cancers, 4 esophageal cancers, 3gallbladder cancers, 4 head-and-neck cancers, 2 kidney cancers, 2 livercancers, 19 lung cancers, 2 NHL, 1 AML, 8 ovarian cancers, 2 prostatecancers, 1 rectum cancer, 4 skin cancers, 2 urinary bladder cancers, 6uterus cancers.

EXAMPLES

Materials and Methods

Patients, Blood and Bone Marrow Samples

Bone marrow mononuclear cells (BMNC) and peripheral blood mononuclearcells (PBMC) from MM patients at the time of diagnosis or at relapsebefore therapy, as well as PBMCs, BMNCs and granulocytes of healthyvolunteers (HV), were isolated by density gradient centrifugation(Biocoll, Biochrom GmbH) and erythrocyte lysis (EL buffer, Qiagen).Informed consent was obtained in accordance with the Declaration ofHelsinki protocol. The study was performed according to the guidelinesof the local ethics committee (142/2013602). Patient characteristics areprovided in table 1. HLA typing was carried out by the Department ofHematology and Oncology, Tubingen, Germany.

Myeloma Cell Lines (MCL)

For HLA ligandome analysis myeloma cell lines (MCLs: U266, RPM18226,JJN3, LP1, MM.1S) were cultured in the recommended cell media (RPM11640,Gibco/IMDM, Lonza) supplemented with 10%/20% fetal calf serum, 100 IU/Lpenicillin, 100 mg/L streptomycin, and 2 mmol/L glutamine at 37° C. and5% CO₂. The MCLs RPM18226, JJN3, MM.1S and LP-1 were obtained from theDepartment of Hematology and Oncology, Tubingen.

Quantification of HLA Surface Expression

HLA surface expression on MM patient and HV bone marrow cells includingCD38⁺CD138⁺ myeloma cells/plasma cells, CD19⁺CD20⁺ B cells, CD3⁺ T cellsand CD34⁺CD38⁻ hematopoietic progenitor cells (HPC) were analyzed usingthe QIFIKIT bead based quantitative flow cytometric assay (Dako)according to manufacturer's instructions as described before [12]. Inbrief, sample were stained with the pan-HLA class I specific monoclonalantibody (mAb) W6/32, HLA-DR specific mAb L243 (produced in house) orIgG isotype control (BioLegend), respectively. Surface marker stainingwas carried out with directly labeled CD138, anti-K, anti-A, CD19, CD20(BioLegend) and CD38, CD3 and CD34 (BD) antibodies. 7-AAD (BioLegend)was added as viability marker immediately before flow cytometricanalysis on a LSR Fortessa (BD).

Isolation of HLA Ligands from Primary Samples and MCLs

HLA class I and II molecules were isolated using standard immunoaffinitypurification as described [44] using the pan-HLA class I specific mAbW6/32, the pan-HLA class II specific mAb Tü39 and the HLA-DR specificmAB L243 (produced in house).

Analysis of HLA Ligands by LC-MS/MS

HLA ligand extracts were analyzed in 5 technical replicates as describedpreviously [13]. In brief, peptide samples were separated by nanoflowHPLC (RSLCnano, ThermoFisher) using a 50 μm×25 cm PepMap RSLC column(Thermo Fisher) and a gradient ranging from 2.4 to 32.0% acetonitrileover the course of 90 min. Eluting peptides were analyzed in an onlinecoupled LTQ Orbitrap XL mass spectrometer (Thermo Fisher) using a top 5CID (collision induced dissociation) fragmentation method.

Database Search and Spectral Annotation

Data processing was performed as described previously [13]. In brief,the Mascot search engine (Mascot 2.2.04; Matrix Science, London, UK) wasimplemented to search the human proteome as comprised in the Swiss-Protdatabase (20,279 reviewed protein sequences, September 2013) withoutenzymatic restriction. Potential mutated HLA ligands were searchedimplementing a database containing the human proteome concatenated withproteins containing single amino acid variants (SAVs) listed in theCOSMIC database (http://cancer.sanger.ac.uk/cosmic/). Only recurrentSAVs described in 2 or more samples of hematological origin wereincluded. Oxidized methionine was allowed as a dynamic modification. Thefalse discovery rate was estimated using the Percolator algorithm [45]and set to 5%. Peptide lengths were limited to 8-12 amino acids for HLAclass I and 12-25 amino acids for HLA class II. Protein inference wasdisabled, allowing for multiple protein annotations of peptides. HLAannotation was performed using SYFPEITHI [46] or an extended in-housedatabase. Experimental validation of peptide identifications and HLAannotations was performed by mass spectrometric and functionalcharacterization of synthetic peptides for a subset of peptides.

Peptide and HLA Peptide Monomer Synthesis

The automated peptide synthesizer EPS221 (Abimed) was used to synthesizepeptides using the 9-fluorenylmethyl-oxycarbonyl/tert-butyl (Fmoc/tBu)strategy [47]. Synthetic peptides were used for validation of LC-MS/MSidentifications as well as for functional experiments. Biotinylatedrecombinant HLA molecules and fluorescent MHC-peptide-tetramers wereproduced as described previously [48].

Amplification of Peptide-Specific T Cells

PBMC from MM patients and HVs were cultured as described previously [12,13]. In brief, for CD8⁺ T-cell stimulation, PBMC were pulsed with 1μg/ml per peptide and cultured for 12 days adding IL-4 and IL-7 on day 0and 1 as well as IL-2 on day 3, 5, 7 and 9. HLA-A*02 (KLFEKVKEV) (SEQ IDNO. 231) and B*07 (KPSEKIQVL) (SEQ ID NO. 232) restricted controlpeptides derived from benign tissues (HV-exclusive HLA ligands) servedas negative control. Peptide-stimulated PBMC were analyzed by ELISPOTassays on day 12. For CD4⁺ T-cell stimulation, culture was performed asdescribed for CD8⁺ T cells except for 2 modifications: pulsing wascarried out with 10 μg/ml of HLA class II peptide and no IL-4 or IL-7was added.

IFNγ ELISPOT Assay

IFNγ ELISPOT assays were carried out as described previously [49]. Inbrief, 96-well nitrocellulose plates (Millipore) were coated with 1mg/ml IFNγ mAb (Mabtech) and incubated over night at 4° C. Plates wereblocked with 10% human serum for 2 h at 37° C. 2.5×10⁵ cells/well ofpre-stimulated PBMC were pulsed with 1 μg/ml (HLA class I) or 2.5 μg/ml(HLA class II) peptide and incubated for 24-26 h. Readout was performedaccording to manufacturer's instructions. PHA was used as positivecontrol. HLA-A*02 (KLFEKVKEV) (SEQ ID NO. 231) and B*07 (KPSEKIQVL) (SEQID NO. 232) restricted control peptides derived from benign tissues(HV-exclusive HLA ligands) served as negative control. Spots werecounted using an ImmunoSpot S5 analyzer (CTL). T cell responses wereconsidered to be positive when >10 spots/well were counted and the meanspot count per well was at least 3-fold higher than the mean number ofspots in the negative control wells (according to the cancerimmunoguiding program (CIP) guidelines [50]).

aAPC Priming of Peptide-Specific T Cells

For the generation of artificial antigen presenting cells (aAPC), 4×10⁶streptavidin-coated polystyrene particles (Bangs Laboratories) per mlwere resuspended in PBE (PBS/BSA/ETDA, Gibco/Sigma Aldrich/Lonza)containing 200 μM biotinylated MHC-peptide monomer and 20 nM anti-humanbiotinylated CD28 antibody and incubated at room temperature for 30 min.After washing, the aAPCs were stored at 4° C. prior to use [51]. CD8⁺ Tcells from MM patients and HV were enriched by positive selection usingmagnetic cell sorting (Miltenyi Biotec). Stimulations were initiated in96-well plates with 1×10⁶ T cells plus 2×10⁵ aAPCs in 200 μl T-cellmedium complemented with 5 ng/ml human IL-12 (PromoKine). 65 U/μl IL-2(R&D Systems) were added on day 5. aAPC stimulation was repeated on day10, for a total of 3 cycles.

Tetramer Staining

The frequency of peptide-specific CD8⁺ T cells was determined on a FACSCanto II cytometer (BD Bioscience) by staining with anti-CD8 (Biolegend)and HLA:peptide-tetramer-PE as described previously [51]. Staining withtetramers containing the CMV pp65 A*02 peptide NLVPMVATV (SEQ ID No.236) served as positive control, tetramers containing irrelevant,non-primed A*02 restricted control peptides as negative controls.Successful priming was considered if frequency of peptide-specific CD8⁺T cells was >0.1% of viable cells and at least 3-fold higher than thefrequency of peptide-specific CD8⁺ T cells in the negative control.

Software and Statistical Analysis

Flow cytometric data analysis was performed using FlowJo 7.2 (Treestar).In-house R and Python scripts were used for the generation of virtualligandomes and definition of virtual TAAs (tumor-associated antigens) inthe analysis of TAA false discovery rates and for the TAA-plateauregression analysis. The standard R heatmap.2 script was used for theunsupervised cluster analysis of HLA ligand source proteins. Graph PadPrism 6.0 (Graph Pad Software) was used for statistical analysis.Statistical analysis of HLA surface expression was based on unpairedt-tests.

HLA Class I Ligand Presentation During Proteasome Inhibitor Therapy.

The inventors quantitatively assessed HLA class I ligand presentationduring proteasome inhibitor therapy. Observed was a considerableplasticity of the HLA class I ligandome after treatment with carfilzomibwith 17.9±1.1% of MM.1S ligands and 11.2±0.7% of U266 ligands (mean ofthree biological replicates±s.d.) showing significant modulation(fold-change ≥4, Po0.01 after Benjamini-Hochberg correction) at t24 hcompared with mock-treated controls. Briefly, Cultured MCLs (MM.1S andU266) and primary myeloma samples were incubated with carfilzomib (100nM, Kyprolis®, available, e.g., from Onyx Pharmaceuticals, Inc.) as anexample for a proteasome inhibitor for a 1-h period, followed by threewashes in PBS (Gibco) and recultured for additional 24 or 48 h. Controlswere incubated with vehicle control (glucose 5%) for 1 h, followed byidentical washing and incubation for 24 or 48 h. Experiments wereconducted in three biological replicates where indicated. The resultsare shown in the following tables.

TABLE 5AMyeloma-associated peptides as detected on MM.1S cells and theirmodulation upon carfilzomib-treatment Myeloma LiTAP HLA SEQ ID NO 24#124#2 24#3 48#1 48#2 48#3 RYLDLFTSF A*24: 02 161 −1 −1 −1  0 −1 −1AFIQAGIFQEF A*23: 01   4 −1  0 −1  0  0 −1 SEFDFFERL C*12: 03 165  0  0 0  0  0 −1 YVFPGVTRL C*12: 03 227  0  0  0 −1  0  0 TFLPFIHTI A*23: 01196  0  0  0  0  0  0 RYFKGPELL A*24: 02 160  0  0  0  0  0  0 RYSPVLSRFA*24: 02 163  0  0  0  0  0  0 RYSTQIHSF A*24: 02 164  0  0  0  0  0  0SYLNSVQRL A*24: 02 192  0  0  0  0  0  0 YYLNEIQSF A*24: 02 228  0  0  0 0  0  0 NEFPVFDEF B*18: 01 128  0  0  0  0  0  0 IPAKPPVSF B*42: 01  76 0  0  0  0  0  0 RPHGGKSL B*42: 01 149  0  0  0  0  0  0 RPQLKGVVLB*42: 01 151  0  0  0  0  0  0 SPALPGLKL B*42: 01 181  0  0  0  0  0  0TPAVGRLEV B*42: 01 200  0  0  0  0  0  0 FAQIISVALI C*12: 03  43  0  0 0  0  0  0 FAYPAIRYL C*12: 03  44  0  0  0  0  0  0 FVFPGELLL C*12: 03 51  0  0  0  0  0  0 VPLPPKGRVL C*12: 03 210  0  0  0  0  0  0LAFPGEMLL A*02: 01 107  0  0  0  0  0  0 APRHPSTNSL B*42: 01  13  0  0 1  0  0  0 RPKAQPTTL B*42: 01 150  0  0  1  0  0  0 TASPLVKSV C*12: 03193  0  0  0  0  1  0 NEVIMTIGF B*18: 01 129  0  0  0  0  1  0 EYGHIPSFA*24: 02  42  1  1  0  0  0  0 TPSSRPASL B*42: 01 202  1  1  0  0  0  0KPQPRPQTL C*12: 03  94  1  1  1  0  0  0 KPRPPQGL B*42: 01  95  1  1  1 1  0  0 IEHPSMSVY B*18: 01  69  1  1  1  0  1  1 VPLTRVSGGAA B*42: 01211  1  1  1  1  1  1 Legend: 24#1-time after carfilzomibtreatment/biological replicate −1 significantly down-modulated 0 notsignificantly/not detected 1 significantly up-modulated

TABLE 5B Myeloma-associated peptides as detected on U266 cells and theirmodulation upon carfilzomib-treatment Myeloma LiTAP HLA SEQ ID NO 24#124#2 24#3 48#1 48#2 48#3 KAMEAASSL B*07: 02  82 −1 −1 −1 −1 −1 −1KPKDPLKISL B*07: 02  93 −1 −1 −1  0 −1 −1 RVFPYSVFY A*03: 01 158 −1  0 0 −1 −1 −1 SRGDFVVEY C*07: 02 190  0 −1  0  0  0 −1 IIFDRPLLY A*03: 01 73  0  0  0  0  0 −1 SVYSPVKKK A*03: 01 191  0  0 −1  0  0  0GEVQDLLVRL B*40: 01  56  0  0  0  0  0  0 KAVNPGRSL B*07: 02  83  0  0 0  0  0  0 SPRLSLLYL B*07: 02 186  0  0  0  0  0  0 ILRDGITAGK A*03: 01 74  0  0  0  0  0  0 RVAKTNSLR A*03: 01 157  0  0  0  0  0  0 TPAVGRLEVB*07: 02 200  0  0  0  0  0  0 SPALKRLDL B*07: 02 180  0  0  0  0  0  0SPRQALTDF B*07: 02 187  0  0  0  0  0  0 AEQEIARLVL B*40: 01   3  0  0 0  0  0  0 KILKPVKKK A*03: 01  88  0  0  0  0  0  0 ALWGRTTLK A*03: 01 10  0  0  0  0  0  0 KPQPRPQTL B*07: 02  94  0  0  0  0  0  0RVNKVIIGTK A*03: 01 159  0  0  0  0  0  0 ILWETVPSM A*02: 01  75  0  0 0  0  0  0 RPGPPTRPL B*07: 02 148  0  0  0  0  0  0 SESLPVRTL B*40: 01167  0  0  0  0  0  0 KLPLPLPPRL A*02: 01  90  0  0  0  0  0  0YLYITKVLK A*03: 01 221  0  0  0  0  0  0 KTEVHIRPK A*03: 01 100  0  0  0 0  0  0 PELGPLPAL B*40: 01 135  0  0  0  0  0  0 RPKAQPTTL B*07: 02 150 0  0  0  0  0  0 FLWDEGFHQL A*02: 01  49  0  0  0  0  0  0 RPFHGVVTSLB*07: 02 147  0  0  0  0  0  0 RQFVVTRTKK A*03: 01 155  0  0  0  0  0  0RPQLKG\NL B*07: 02 151  0  0  0  0  0  0 IESHPDNAL B*40: 01  70  0  0  0 0  0  0 REEGTPLTL B*40: 01 141  0  0  0  0  0  0 GEVAPSMFL B*40: 01  55 0  0  0  0  0  0 SPYLRPLTL B*07: 02 189  0  0  0  0  0  0 KPSTKALVLB*07: 02  97  0  0  0  0  0  0 KLSSLIILM A*02: 01  92  0  0  0  0  0  0LPPPPHVPL B*07: 02 115  0  0  0  0  0  0 GETAFAFHL B*40: 01  54  0  0  0 0  0  0 LLFPYILPPK A*03: 01 111  0  0  0  0  0  0 IPAKPPVSF B*07: 02 76  0  0  0  0  0  0 GPRPITQSEL B*07: 02  61  0  0  0  0  0  0TPSSRPASL B*07: 02 202  0  0  0  0  0  0 RPRPPVLSV B*07: 02 154  0  0  0 0  0  0 KEGLILPETL B*40: 01  87  1  1  0  0  0  0 FAYPAIRYL A*02: 01 44  0  0  0  1  0  1 FVFPGELLL A*02: 01  51  1  1  0  0  0  0APFQGDQRSL B*07: 02  11  0  1  1  0  1  0 KPRPPQGL B*07: 02  95  0  0  0 1  1  1 APRHPSTNSLL B*07: 02  14  0  1  0  1  1  0 APRHPSTNSL B*07: 02 13  0  1  0  1  1  0 Legend: 24#1-time after carfilzomibtreatment/biological replicate −1 significantly down-modulated 0 notsignificant/not detected 1 significantly up-modulated

The MM.1S model was then used to longitudinally track the abundances ofthe 14/31 myeloma peptides for which quantitative information wasavailable across all time points and conditions. For the majority ofthese targets (10/14, 71.4%), we observed a peak in modulation at t24 hfollowed by a gradual decline toward baseline levels at t48 h. Only 4/14peptides (28.6%) showed persistent modulation even at t48 h, with threeof them showing progressive down-modulation after treatment.

Amongst others, SEQ ID NO: 42: showed a significant up-modulation onMM.1s cells 24 h after carfilzomib treatment in 2/3 biologicalreplicates. In contrast, no significant modulation upon carfilzomibtreatment was found on MM.1S for SEQ ID NO: 107 and SEQ ID NO: 228.

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1. A method of eliciting an immune response in a patient who has cancer,comprising administering to the patient a composition comprising apopulation of activated T cells that selectively recognize cancer cellsthat present a peptide consisting of the amino acid sequence selectedfrom the group consisting of from SEQ ID NO: 1 to SEQ ID NO: 32 and fromSEQ ID NO: 34 to SEQ ID NO: 228, wherein the activated T cells areproduced by contacting T cells with the peptide loaded onto a humanclass I MHC molecule expressed on the surface of an antigen-presentingcell for a period of time sufficient to activate the T cells, whereinsaid cancer is selected from the group of lung cancer, kidney cancer,brain cancer, stomach cancer, colon or rectal cancer, liver cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma (MCC),melanoma, ovarian cancer, esophageal cancer, urinary bladder cancer,endometrial cancer, gall bladder cancer, and bile duct cancer.
 2. Themethod of claim 1, wherein the T cells are autologous to the patient. 3.The method of claim 1, wherein the T cells are obtained from a healthydonor.
 4. The method of claim 1, wherein the T cells are obtained fromtumor infiltrating lymphocytes or peripheral blood mononuclear cells. 5.The method of claim 1, wherein the activated T cells are expanded invitro.
 6. The method of claim 1, wherein the peptide is in a complexwith the class I MHC molecule.
 7. The method of claim 1, wherein theantigen presenting cell is infected with recombinant virus expressingthe peptide.
 8. The method of claim 7, wherein the antigen presentingcell is a dendritic cell or a macrophage.
 9. The method of claim 5,wherein the expansion is in the presence of an anti-CD28 antibody andIL-12.
 10. The method of claim 1, wherein the population of activated Tcells comprises CD8-positive cells.
 11. The method of claim 1, whereinthe contacting is in vitro.
 12. The method of claim 1, wherein thecomposition further comprises an adjuvant.
 13. The method of claim 12,wherein the adjuvant is selected from the group consisting of anti-CD40antibody, imiquimod, resiguimod, GM-CSF, cyclophosphamide, Sunitinib,bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides andderivatives, poly-(I:C) and derivatives, RNA, sildenafil, andparticulate formations with poly(lactide co-glycolide) (PLG), virosomes,interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, andIL-23.
 14. The method of claim 1, wherein the immune response comprisesa cytotoxic T cell response.
 15. A method of killing cancer cells,comprising performing the method of eliciting an immune response ofclaim 1, wherein the cancer cells are killed.
 16. The method of claim15, wherein the immune response comprises a cytotoxic T cell response.